Summary

We have previously shown that aPKC interacts with cell polarity proteins
PAR-3 and PAR-6 and plays an indispensable role in cell polarization in the
C. elegans one-cell embryo as well as in mammalian epithelial cells.
Here, to clarify the molecular basis underlying this aPKC function in
mammalian epithelial cells, we analyzed the localization of aPKC and PAR-3
during the cell repolarization process accompanied by wound healing of MTD1-A
epithelial cells. Immunofluorescence analysis revealed that PAR-3 and
aPKCλ translocate to cell-cell contact regions later than the formation
of the primordial spot-like adherens junctions (AJs) containing E-cadherin and
ZO-1. Comparison with three tight junction (TJ) membrane proteins, JAM,
occludin and claudin-1, further indicates that aPKCλ is one of the last
TJ components to be recruited. Consistently, the expression of a
dominant-negative mutant of aPKCλ (aPKCλkn) in wound healing
cells does not inhibit the formation of the spot-like AJs; rather, it blocks
their development into belt-like AJs. These persistent spot-like AJs in
aPKCλ-expressing cells contain all TJ membrane proteins and PAR-3,
indicating that aPKC kinase activity is not required for their translocation
to these premature junctional complexes but is indispensable for their further
differentiation into belt-like AJs and TJs. Cortical bundle formation is also
blocked at the intermediate step where fine actin bundles emanating from
premature cortical bundles link the persistent spot-like AJs at apical tips of
columnar cells. These results suggest that aPKC contributes to the
establishment of epithelial cell polarity by promoting the transition of
fibroblastic junctional structures into epithelia-specific asymmetric
ones.

Introduction

In response to an external cue, cells autonomously generate an asymmetric
distribution of intracellular components, thereby producing functional
asymmetry within themselves both during development and as an adult. The
establishment and maintenance of this `cell polarity' is an essential feature
of all eukaryotic cells and is critical for the integrity of the organism.
Recent studies have made great progress in revealing the molecular basis of
this cell polarity by identifying many sets of polarity proteins and their
interactions (
Bilder, 2001;
Ohno, 2001). One of the most
essential sets of polarity proteins is an evolutionarily conserved molecular
complex, the aPKC-PAR system, which plays a critical role in the establishment
of cell polarity in different organisms and cell types
(
Rose and Kemphues, 1998;
Suzuki et al., 2001;
Wodarz et al., 2000).

PAR (partitioning-defective) proteins, PAR-1 to PAR-6, were first
identified as proteins, mutations of which led to loss of the
anterior-posterior cell polarity of the C. elegans one-cell embryo
(
Guo and Kemphues, 1996). The
finding that mammalian PAR-3 (ASIP) specifically binds to atypical PKC (aPKC)
leads us to further reveal that C. elegans aPKC (PKC-3) interacts
with PAR-3 and also plays an indispensable role in cell polarization of the
C. elegans one-cell embryo (
Izumi
et al., 1998;
Tabuse et al.,
1998). We demonstrated recently that in mammalian epithelial
cells, aPKC interacts with not only PAR-3 but also PAR-6 and plays a critical
role in the formation of the apical-basal polarity
(
Suzuki et al., 2001;
Yamanaka et al., 2001). In
those studies, we overexpressed a dominant-negative mutant of one of the
mammalian aPKC isoforms, aPKCλ, in MDCK epithelial cells and found that
the mutant inhibits the development of the tight junction (TJ) structures as
well as the establishment of asymmetric distribution of membrane proteins only
when cell-cell junctions are reset by calcium switch treatment. These results
indicate that aPKC activity is required for the development but not the
maintenance of epithelial polarity, although the molecular targets of aPKC are
still unknown.

One of the interesting features of the cell polarity proteins revealed so
far is that they themselves asymmetrically localize underneath the restricted
regions of the plasma membrane, and this asymmetric submembranous localization
is critical for their function (
Bilder,
2001;
Ohno, 2001;
Rose and Kemphues, 1998). For
example, the localization of aPKC, PAR-3 and PAR-6 is gradually restricted to
the anterior periphery of the C.elegans one-cell embryo in
response to sperm entry, whereas PAR-1 and PAR-2 are restricted to the
posterior periphery during the development of cell polarity
(
Rose and Kemphues, 1998).
Defects in one of the three proteins, aPKC, PAR-3 or PAR-6, result in the
disruption of the asymmetric localization of all other PAR proteins. On the
other hand, during the asymmetric division of neuroblasts in the
Drosophila embryo, basal crescent localization of neuronal
determinants Miranda and Prospero is directed by the apically localized
Inscuteable (
Doe and Bowerman,
2001). Interestingly, recent findings indicate that the correct
localization of Inscuteable depends on the apical localization of the
Drosophila aPKC-PAR complex, which is inherited from the epithelium
from which neuroblast delaminates (
Ohno,
2001;
Wodarz et al.,
1999). Considering that all of these polarity proteins are not
membrane proteins, these results suggest that the establishment of asymmetric
submembranous structures to which these determinants anchor is essential for
cell polarity. However, the molecular basis of this putative submembrane
structure is not known for the C. elegans or Drosophila
embryonic cells. In this sense, the results from mammalian epithelial cells
provided very important clues to address this issue: in these cells, aPKC,
PAR-3 and PAR-6 are asymmetrically localized to TJ, the specialized cell-cell
junctional structure in the most apical region of the basolateral membrane
(
Dodane and Kachar, 1996;
Izumi et al., 1998).
Furthermore, the cytoplasmic tail of a TJ membrane protein, JAM, interacts
with the first PDZ domain of PAR-3, suggesting that JAM is a strong candidate
for the anchoring partner of the aPKC-PAR complex at TJ
(
Ebnet et al., 2001;
Itoh et al., 2001). Our recent
results on the inhibitory effect of the dominant-negative mutant of
aPKCλ (aPKCλkn) further indicated that the suppression of aPKC
activity resulted in the disruption of the submembranous structure, that is,
TJ to which the aPKC-PAR complex itself is asymmetrically localized
(
Suzuki et al., 2001).
Considering that the formation of the epithelia-specific junctional structures
including TJ, adherens junctions (AJs) and desmosomes, which tightly couple
with the development of asymmetric cytoskeletal structures, represents the
development of submembranous asymmetric structures in epithelial cells
(
Denker and Nigam, 1998;
Yeaman et al., 1999), these
results suggest an intriguing possibility that aPKC primarily regulates the
development of the epithelia-specific junctional structures of epithelial
cells and thus contributes to the development of the apico-basal polarity.

TJ biogenesis has been suggested to be induced by cell-cell adhesion
mediated by E-cadherin (
Gumbiner et al.,
1988), but the molecular mechanism underlying this process is
still unclear. However, by analyzing the wound healing process of a mouse
epithelial cell line, MTD1-A cells, Tsukita and co-workers have provided
evidence that epithelial junctional formation can be dissected into multiple
steps proceeding in a sequential manner during the cell polarization process
(
Ando-Akatsuka et al., 1999;
Yonemura et al., 1995). On the
basis of immunofluorescence analysis, they demonstrated that at the initial
phase of cell polarization, fibroblastic spot-like AJs containing E-cadherin
as well as ZO-1 are formed as a nascent junctional complex
(
Vasioukhin et al., 2000;
Yonemura et al., 1995).
Thereafter, as epithelial polarization progresses, ZO-1 dissociates from
E-cadherin, which separately forms the epithelia-specific belt-like AJ, and
gradually colocalizes with occludin at cell-cell contact sites to form TJs
(
Ando-Akatsuka et al., 1999).
Here, by combining the same experimental system and the dominant-negative
mutant of aPKCλ used previously
(
Suzuki et al., 2001), we
attempted to clarify how aPKCλkn inhibits TJ formation during
epithelial cell polarization. Our results indicate that aPKC is recruited
after the establishment of the initial spot-like AJ complex to which not only
E-cadherin-catenins but also several TJ components such as JAM, occludin and
claudin-1 are transiently recruited and contributes to the further development
of this premature junctional complex into epithelia-specific structures in
which belt-like AJs, TJs, are asymmetrically segregated.

Materials and Methods

Cell culture and wounding

Mouse epithelial cells, MTD1-A cells
(
Enami et al., 1984), were
cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10%
fetal calf serum. For the wound healing assay, the cells were plated on
coverslips at a density of 2.5×105 cells/cm2, and
the next day, the resultant confluent monolayers were scratched manually with
an 18G needle as described previously
(
Ando-Akatsuka et al., 1999).
Wounded regions were allowed to heal for 6-30 hours prior to
immunofluorescence analysis.

Adenovirus infection

The adenovirus vectors carrying cDNA encoding LacZ or aPKCλkn have
been described previously (
Suzuki et al.,
2001;
Yamanaka et al.,
2001). For adenovirus infection, MTD1-A cells were seeded on
coverslips in 24-well plates at a density of 1.25×105
cells/cm2 1 day before infection, as described previously
(
Suzuki et al., 2001). After 2
hours of preincubation in low calcium medium containing 5% FCS and 3 μM
Ca2+ (
Stuart et al.,
1994), the cells were incubated for 2 hours with 150 μl of the
appropriate virus solution diluted to 3×108 pfu/ml in LC
medium. Cells were washed two times with PBS and further cultured in normal
growing medium overnight before wounding.

Confocal immunofluorescence microscopy

At an appropriate time after wounding, cells were fixed with 2%
formaldehyde in PBS for 15 minutes at room temperature. After washing twice
with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10
minutes at room temperature. The cells were then washed and soaked in blocking
solution (PBS containing 10% calf serum) overnight at 4°C. Antibody
incubations were performed at 37°C for 45 minutes in buffer containing 10
mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.01% (v/v) Tween 20 and 0.1% (w/v) BSA.
The secondary antibodies used were Alexa488-conjugated goat anti-rabbit IgG
(Molecular Probes Inc., Eugene, OR), Cy3-conjugated goat anti-mouse IgG,
Cy3-conjugated goat anti-rabbit IgG or Cy3-conjugated goat anti-rat IgG
(Amersham Corp., Arlington Heights, IL). To stain F-actin,
rhodamine-phalloidin (Molecular Probes) was used in place of the secondary
antibodies. Coverslips were mounted using Vectashield (Vector Laboratories,
Burlingame, CA) and examined under a fluorescence microscope equipped with a
confocal system (μRadiance, Bio-Rad Laboratories, Hercules, CA). An
oil-immersion objective lens (Plan APOCHROMAT ×63, NA 1.40) (Nikon), and
argon and red diode lasers were used. Conventional images were composed of
512×512 pixels. Usually, about 30 optical sections covering the basal to
the apical region of cells were taken with an interval of 0.4 μm, and all
images were projected unless indicated otherwise.

Results

aPKC and PAR-3 are recruited into cell-cell contact regions after
establishment of spot-like AJ during the wound healing process of MTD-1A
cells

MTD1-A cells derived from a mouse mammary tumor
(
Enami et al., 1984) are
useful for analyzing the epithelial cell polarization process, because they
show (1) less frequent overriding of the cell peripheries during the
establishment of cell polarity, and (2) highly synchronous formation of
cell-cell contact regions (
Yonemura et
al., 1995). The combination of this cell line and the
wound-healing assay provides good resolution for analyzing the epithelial
junctional formation process step by step
(
Ando-Akatsuka et al., 1999).
We first attempted to clarify at which steps aPKCλ and PAR-3 are
recruited into the cell-cell junctional region. When a confluent monolayer of
MTD-1A cells was manually scratched with a needle, nascent cell-cell contacts
appeared around 6-8 hours after wounding. At initial stages of wound closure,
encountering cells formed many spot-like AJs positive for E-cadherin as well
as ZO-1 at the tip of very thin membrane protrusions
(
Ando-Akatsuka et al., 1999;
Yonemura et al., 1995).
Immunofluorescence analysis using anti-PAR-3 or anti-aPKCλ antibody
revealed that most of these spot-like AJs identified with the anti-ZO-1
antibody were negative for PAR-3 and aPKCλ (small arrows in
Fig. 1A,B). Even at later
stages when ZO-1-positive structures had gradually fused and became continuous
short fragments, both proteins showed retarded recruitment into these
junctions (small arrows in
Fig.
1A,B, middle and right panels). Indirect comparison of the timing
of the recruitment of PAR-3 and aPKCλ into cell-contact regions
relative to that of ZO-1 suggested that aPKCλ was recruited later than
PAR-3, although the lack of available antibodies did not allow us to confirm
it directly. Nevertheless, these results indicate that PAR-3 and aPKCλ
are recruited into the junctional complex after the formation of the initial
spot-like AJs containing E-cadherin and ZO-1.

aPKCλ and PAR-3 translocate to cell-cell junctional regions after
the establishment of spot-like AJs. (A,B) Confluent MTD1-A monolayers were
scratched with a needle, cultured for 6 hours (left panels), 9 hours (middle
panels) and 12 hours (right panels) and then doubly stained with PAR-3 pAb and
ZO-1 mAb (A) or aPKCλ pAb and ZO-1 mAb (B), as indicated. Each panel
represents a projected view of confocal optical sections (0.4 μm) collected
from the apical to the basal region of the cells, although colocalization of
the proteins was confirmed in a single section. (The panels in the following
figures are similarly obtained unless otherwise mentioned.) Large arrowheads
indicate the direction of the wounds. Note that many ZO-1-positive spot-like
AJs are negative for PAR-3 as well as aPKCλ staining. In contrast with
PAR-3, aPKCλ signal is often lacking on the ZO-1-positive continuous
junctions, Bars, 10 μm.

aPKCλ is recruited last to TJs

The TJ membrane proteins occludin and JAM colocalize with ZO-1 and
E-cadherin in the spot-like AJs prior to forming continuous TJ structures
(
Ando-Akatsuka et al., 1999;
Ebnet et al., 2001). Therefore,
the results in
Fig. 1 suggest
that aPKC and PAR-3 are recruited into cell-contact regions later than these
TJ membrane proteins. In fact, we demonstrated by double staining analysis
that many JAM-positive spot-like AJs observed in intermediate stages of the
wound healing of MTD1-A cells were negative for PAR-3
(
Ebnet et al., 2001). Again,
owing to the lack of appropriate antibodies, we could not perform similar
experiments for the other TJ membrane proteins, occludin as well as claudin-1.
However, during indirect comparison between these TJ proteins and PAR-3/aPKC
with respect to junctional recruitment, we unexpectedly found that claudin-1
is recruited to the junctions considerably later than JAM and occludin and as
late as aPKCλ and PAR-3. As demonstrated previously, JAM almost
completely colocalized with the spot-like AJs positive for ZO-1 formed at the
very early stage of wound healing (
Fig.
2A top panels). The occludin signal was also detected in most of
the nascent spot-like AJs, although its signal on the ZO-1-positive dot-like
AJs tended to be weaker than that of JAM (arrows in
Fig. 2A, middle panels). On the
other hand, many ZO-1-positive dot-like or fragmental structures at nascent
cell-cell contacts showed a weak or negative signal for claudin-1 (arrows in
Fig. 2A, bottom panels). Even
relatively developed junctions more than 3 μm long were frequently negative
for claudin-1 staining. These results suggest that claudin-1 is recruited into
the junctional area after the establishment of the dot-like AJs. This was
further confirmed by the direct comparison between doubly stained JAM and
claudin-1 (
Fig. 2B, top
panels); again, many JAM-positive junctional structures of 3 μm length were
often negative for claudin-1 staining. Considering the similarity of the
immunostaining pattern of claudin-1 to those of PAR-3 and aPKCλ in
nascent junctional areas (
Fig.
1,
Fig. 2B bottom
panels), these results indicate that TJ components can be subdivided into at
least two groups in terms of junctional recruitment, and the polarity
proteins, aPKC and PAR-3, as well as claudin-1 belong to the same group whose
recruitments is rather late.

Claudin-1 is a unique TJ component that is recruited into junctional
regions as late as PAR-3 and aPKCλ are. The recruitment of TJ
components into the cell-cell junctional regions of wound healing cells was
examined by double immunostaining at intermediate stages of wound closure. In
A, three TJ membrane proteins, JAM, occludin and claudin-1, were compared with
ZO-1, whereas, in B, claudin-1 and PAR-3 were compared with JAM, as indicated.
The combination of used antibodies (rat JAM mAb, rabbit occ pAb, rabbit
claudin-1 pAb, mouse ZO-1 mAb and anti-PAR-3 pAb) is indicated in the panels,
and merged views are also presented on the right. The direction of the wounds
is indicated by large arrowheads, whereas ZO-1- or JAM-positive spot-like AJs,
which showed negative or weak staining by corresponding other TJ proteins, are
indicated by small arrows. Small arrowheads indicate occludin- or
claudin-1-positive, but ZO-1 negative, granular structures sometimes observed
in cytoplasm. Note that many ZO-1- or JAM-positive dot-like AJs are negative
for claudin-1. Bars, 10 μm.

Overexpression of a dominant-negative mutant of aPKCλ inhibits
the development of spot-like AJs into epithelia-specific belt-like AJs

Previously, by using the adenovirus gene-transfer technique, we
demonstrated that overexpression of a kinase-negative mutant of aPKCλ
or ζ (aPKCkn) disturbs the junctional formation of MDCK II cells observed
in the re-polarization process after a calcium switch treatment through their
dominant-negative activity against endogenous aPKC (λ or ζ) kinase
activity (
Suzuki et al.,
2001). To address the question of which steps of the epithelial
junctional formation pathway are blocked by aPKCkn, we applied this
aPKCλ mutant to the wound-healing assay using MTD-1A. When a confluent
monolayer of MTD1A cells (2.5×105 cells/cm2) was
infected with adenovirus vector at MOI 600, aPKCλkn was expressed in
approximately 90% of the cells showing heterogeneous expression levels
(
Fig. 3A, lower left panel).
Similar to the situation shown for MDCK II cells in previous work
(
Suzuki et al., 2001),
junctional structures monitored by ZO-1 staining were hardly affected by the
expression of aPKCλkn unless the infected cells were subjected to
regeneration of cell-cell adhesion by a calcium switch treatment (data not
shown) (see ZO-1 staining at cell-cell boundaries in non-wounded regions of
aPKCλkn-expressing cells in
Fig.
3A, lower right panel). When monolayers of MTD1-A cells expressing
aPKCλkn or LacZ were wounded, they showed apparently normal wound
closure within 6-10 hours under phase-contrast microscope observation (data
not shown). In LacZ-expressing cells, continuous ZO-1 staining was completely
restored in the healed regions (
Fig.
3A, upper right panel). This was also the case when nPKCϵkn
was used as a negative control instead of LacZ (data not shown). However, in
cells expressing aPKCλkn, the completion of TJ formation monitored by
ZO-1 was significantly inhibited in cells burying the wound
(
Fig. 3A, lower panels). Closer
inspection demonstrated that in cells that participated in burying the wound,
ZO-1 staining was broadly observed in dot-like structures or in very short
fragments at cell-cell borders, and this staining pattern did not change even
after the wound was completely healed (30 hours after wounding,
Fig. 3B). These cells also
exhibited aberrant E-cadherin staining that revealed dot-like discontinuous
structures instead of belt-like AJs as observed in LacZ-expressing cells.
Comparison of ZO-1 and E-cadherin staining in a single confocal plane
confirmed that many ZO-1-positive dot-like AJs, if not all, are also positive
for E-cadherin (
Fig. 4, top
panels). It was further demonstrated that α-catenin and nectin also
showed colocalization with ZO-1 in these structures with higher frequency
(
Fig. 4, middle and bottom
panels). Together with the fact that many F-actin bundles are running into
these dot-like structures (see below), these results indicate that the
ZO-1-positive dot-like structures induced by aPKCλkn expression are
structurally identical to the spot-like AJs observed during the normal wound
healing process, which appear only in newly formed cell-cell contacts between
encountering cells in the first row of the wound margin. Taken together, the
results in Figs
3 and
4 suggest that the inhibition
of aPKCλ kinase activity does not suppress the formation of the
primordial spot-like AJ complexes; rather, it blocks their development into
the epithelia-specific belt-like AJs.

Ectopic expression of the dominant-negative mutant of aPKCλ
(aPKCλkn) inhibited the regeneration of TJ in the wound-healing area.
Confluent monolayers of MTD1-A cells were infected with adenovirus vectors
carrying LacZ or aPKCλkn cDNA, and the ectopic protein expression was
induced in the normal growth media for 18 hours. The wound-healing assay was
performed as described in the legend to
Fig. 1 (large arrowheads
indicate the direction of the wounds) and, 10 hours (A) or 30 hours (B) after
wounding, cells were fixed and doubly stained with anti-aPKCλ pAb and
anti-ZO-1 mAb (A) or anti-ZO-1 pAb and anti-E-cadherin mAb (B). As shown in A,
although the expression levels are virtually heterogenous, aPKCλkn was
expressed in almost 90% of the cells at levels higher than the endogenous one
(under the photographic conditions used here, fluorescence signals of
endogenous aPKCλ cannot be detected). Note that when aPKCλkn was
expressed, ZO-1 staining disappeared in cells burying the wound, although the
completion of wound healing was confirmed by phase-contrast microscopy (data
not shown) (see aPKCλ staining in cells burying the wound). Enlarged
views in B revealed that ZO-1 is broadly localized in dot-like structures in
the wound-healing region of aPKCλkn-expressing cells, where E-cadherin
staining also displayed rough, discontinuous appearance. Bars, 100 μm (A)
or 20 μm (B).

Overexpression of aPKCλkn inhibited the development of spot-like AJs
into belt-like AJs. aPKCλkn-expressing cells were subjected to
wounding, fixed 30 hours after the wounding and doubly immunostained by the
antibodies indicated. Large arrowheads indicate the direction of the wounds.
The projected views of confocally obtained optical sections from the apical to
the basal regions of the cells, except for merged views which were made from
appropriate single confocal sections to strictly examine the co-localization
of the two proteins (green: AJ proteins, red: ZO-1), are shown. Three AJ
proteins, E-cadherin, α-catenin and nectin, were colocalized to some of
these persistent ZO-1-positive spot-like structures (small arrows in the
merged view), suggesting that these correspond to the spot-like AJs that could
not develop into belt-like AJs. The structures were located at the apex of the
lateral membrane (see
Fig. 5B),
and E-cadherin and α-catenin also distributed diffusely in the lateral
membranes (small arrowheads in middle panels). Bars, 20 μm.

The cortical F-actin bundle formation process is trapped in an
intermediate state in aPKCλkn-expressing cells

Epithelia-specific belt-like AJs are characterized by their ability to
align with a thick actin cable called the cortical (peripheral) bundle
(
Hirano et al., 1987).
Consistent with the results in
Fig.
4, rhodamine-phalloidin staining revealed that the formation of
the cortical F-actin bundle was also inhibited in wound-healing cells
expressing aPKCλkn (
Fig.
5A, middle and bottom panels). Instead, many stress fiber-like
F-actin bundles were running into the persistent spot-like AJs positive for
ZO-1 in these cells. Significantly, in some cells, a loosely bundled F-actin
cable was observed to run circularly underneath the membrane, from which many
fine F-actin bundles emanated into the spot-like AJs. It should be noted that
this distribution pattern of F-actin is strikingly similar to the intermediate
state of F-actin reorganization observed at intermediate stages of the normal
polarization process of MTD1-A cells, which finally develops into cortical
bundles (
Yonemura et al.,
1995). These results strongly support the notion that aPKC kinase
activity plays a critical role in this normal process of belt-like AJ
development. In addition, they also suggested that the fusion of the premature
spot-like AJ into belt-like AJs is coupled to F-actin reorganization so
tightly, as though they were either side of the same coin, that they cannot
proceed separately.

Development of the cortical bundle of F-actin was blocked in an
intermediate step in aPKCλ-expressing cells burying the wound.
aPKCλkn-expressing cells burying the wound were doubly stained with
rhodamine-phalloidin (red) and anti-ZO-1 antibody (green) 10 hours (A) or 30
hours (B and C) after wounding. Large arrowheads indicate the direction of the
wounds. In A, the results for LacZ-expressing cells are also shown for
comparison (top panels). Shown are projected views of confocally obtained
optical sections covering apical regions (A, and top panels in B), single
confocal section at basal, intermediate, or apical region (C) or z-sectional
views crossing a spot-like incomplete junctional structure (small arrowhead;
bottom panels of B). aPKCλkn-expressing cells do not complete cortical
bundle formation of F-actin, showing stress fiber-like F-actin bundles linking
ZO-1-positive spot-like AJs. In some cells, the prototype of cortical loose
bundles of F-actin was observed, from which short F-actin fibers emanate into
the spot-like AJs (right panels in A). Note that this kind of F-actin
organization is formed in the apical surface of cells independently of the
basal stress fibers (B,C), and cells with spot-like AJs also develop in height
to a level comparable to that of surrounding cells with complete TJs. Bars, 10μ
m.

Interestingly, confocal z-sectional analysis revealed that
aPKCλkn-expressing cells burying the wound also develop a columnar
shape with a height comparable to that of surrounding cells, and the spot-like
AJs reside at the apical tip of the lateral membranes (small arrowhead in
Fig. 5B). In fact, besides
being distributed on the dot-like AJs, E-cadherin and α-catenin also
showed broad distribution on the lateral membrane (small arrowheads in
Fig. 4, top and middle panels),
suggesting that asymmetric domain formation in the lateral membrane occurred
even in aPKCkn-expressing cells lacking mature belt-like AJs. In these cells,
the premature cortical bundle structures linking the spot-like AJs were formed
underneath the apical membrane independently of the basal stress fibers
(
Fig. 4B,C). Therefore, these
results indicate that in aPKCλkn-expressing cells, F-actin
reorganization to restore cortical bundles and the epithelia-specific columnar
shape proceeds to some extent during the re-epithelialization process, but the
final step involving connection of the F-actin cortical bundles closely to
fused belt-like AJs is inhibited completely in these cells.

TJ membrane proteins and PAR-3 are trapped in the persistent
spot-like AJs in aPKCkn-expressing cells

As shown in
Fig. 6A, almost
all of the ZO-1-positive spot-like AJs in aPKCλkn-expressing
wound-healing cells were positive for JAM and occludin. Interestingly,
claudin-1 also showed almost complete colocalization with ZO-1 in the dot-like
structures induced by aPKCλkn, although during the normal wound healing
process, it is recruited into the junctional area rather late and is thus
hardly detected in the dot-like AJs (
Fig.
2). These results strongly reinforce the idea that the dot-like
AJs are intermediate structures for epithelial junctional development to which
TJ components including claudin-1 can be transiently recruited. Furthermore,
they also indicate that aPKCλ kinase activity is not required for this
translocation. Rather, aPKCλ plays a critical role in the subsequent
segregation step of the junctional proteins in these primordial AJ structures
into mature belt-like AJs and TJs.

Three TJ membrane proteins were also trapped in the persistent spot-like AJ
of aPKCλkn-expressing cells. aPKCλkn-expressing cells were
subjected to wounding, fixed 30 hours after the wounding and doubly
immunostained with pAbs against TJ proteins or PAR-3 and ZO-1 mAb, as
indicated. Shown are enlarged views of cells burying the wound or their merged
views. JAM, occludin and claudin-1 are all colocalized with ZO-1 in the
persistent spot-like AJs (A). As shown in B, a substantial number of the
spot-like AJs induced by aPKCλkn are also positive for PAR-3, although
the extent of the colocalization is relatively low (small arrows indicate
difference in staining between PAR-3 and ZO-1). Bars, 10 μm.

We next examined the localization of PAR-3 in aPKCλkn-expressing
wound-healing cells and found that a substantial part of the persistent
dot-like AJs was positive for PAR-3 (
Fig.
6B). Therefore, PAR-3 translocation to the spot-like AJs can also
occur without aPKCλkn activity. This is consistent with the indirect
observation that PAR-3 translocates to the spot-like AJs before the
recruitment of aPKC into the junctional regions
(
Fig. 1). However, it should
also be noted that in contrast with other TJ components, many ZO-1-positive
spot-like AJs show weak or negative staining for PAR-3 even 30 hours after
wounding (see small arrows in
Fig.
6B, right panel). This may indicate that the translocation or
stability of PAR-3 at the spot-like AJs partially depends on aPKC
activity.

Discussion

Spot-like structures (also called puncta) positive for E-cadherin are
transiently formed as an intermediate during the development of the
epithelia-specific continuous cell-cell junctions
(
Adams et al., 1998;
Vasioukhin et al., 2000;
Yonemura et al., 1995).
Colocalization of α-actinin, vinculin, as well as ZO-1 and termination
of prominent F-actin bundles at these structures have established that these
nascent AJ structures correspond to the spot-like AJs observed in fibroblastic
cells (
Vasioukhin et al.,
2000;
Yonemura et al.,
1995). Using the wound healing assay of MTD1-A cells,
Ando-Akatsuka et al. have extended these observations by showing that ZO-1
colocalizes with E-cadherin in the spot-like very early AJ structures prior to
the recruitment of occludin, but as junctional formation proceeds, ZO-1
dissociates from E-cadherin to form TJ with occludin
(
Ando-Akatsuka et al., 1999).
These results have created the idea that the epithelial junctional formation
can be divided into two phases: first is the formation of the fibroblastic
spot-like AJs at the nascent cell-cell contacts, into which several AJ and TJ
proteins are transiently and sequentially recruited, and second is the
differentiation of the primordial adhesion complex into epithelia-specific
well segregated junctional structures such as belt-like AJs and TJs.

In this work, we reinforced this idea by finding that the overexpression of
aPKCλkn results in the blockage of development of the epithelial
junctional structure at the formation of spot-like AJs. The present results
indicate that aPKC activity is not required for the first step of junctional
development, that is, the recruitment of junctional proteins into contact
sites, but is indispensable for the second step to reconstruct the nascent
junctional complex into epithelia-specific mature junctional structures.
Significantly, the persistent spot-like AJs in aPKCλkn-expressing cells
contain multiple junctional membrane proteins, such as E-cadherin, nectin,
JAM, occludin and claudin-1, confirming that the spot-like AJs are structures
in which many junctional proteins gather together prior to their subsequent
segregation. These membrane proteins are considered to assemble in the
structures by interacting directly or indirectly (through peripheral protein
such as catenins for E-cadherin) with various peripheral scaffold proteins
containing PDZ domains, such as ZO-1, afadin and MUPP1, which can associate
with the cytoplasmic regions of the multiple membrane proteins
(
Ebnet et al., 2000; Hamazaki
et al., 2001;
Itoh et al.,
1999;
Itoh et al.,
2001). Therefore, these results raise an intriguing possibility
that aPKC is responsible for modification of the interactions between
junctional membrane proteins and scaffold proteins or between membrane
proteins by themselves, thereby promoting the transition of junctional
structures from the first step to the second one. This is consistent with
another observation in this study that aPKC is one of the later TJ components
with respect to junctional recruitment.

Of course, in addition to modifying junctional proteins, aPKC may affect
junctional formation by interfering with other events required for the
development of belt-like AJs and TJs. In this sense, it should be noted that
Vasioukhin et al. have suggested that an antiparallel pair of filopodia, at
the tip of which the spot-like AJs (or puncta) are formed, physically draws
the two cell surfaces together, thereby extending the zone of cell-cell
contact (
Vasioukhin et al.,
2000). They further demonstrated that actin polymerization at the
tip of the protrusions plays an active role in extending the cell-cell contact
area. Therefore, the regulation of F-actin polymerization and reorganization
may be another target of aPKC phosphorylation to promote belt-like AJ
formation. In fact, we observed that cortical bundle formation is blocked in
aPKCλkn-expressing cells at an intermediate stage
(
Fig. 5)
(
Yonemura et al., 1995).
However, we also observed that even the cells expressing aPKCλkn
lacking continuous junctional structures increased their heights to a level
(>10 μm) comparable to those of cells in non-wounded areas. The
premature spot-like AJs and the premature cortical bundles are localized at
the apical tip of the lateral membranes, at which neighboring cells are in
close contact. Therefore, these results indicate that the cells expressing
aPKCλkn can reorganize the actin filament architecture to some extent
and physically draw neighboring cell surfaces together to assume a thick
epithelia-specific columnar shape. Therefore, the incomplete cortical bundle
formation of F-actin may be a secondary consequence of the inhibition of the
fusion of the spot-like AJs into belt-like ones.

aPKC plays an indispensable role in the establishment of cell polarity not
only in the C. elegans and Drosophila early embryo but also
in mammalian epithelial cells by forming an evolutionarily conserved protein
complex with PAR-3 and PAR-6. We have previously demonstrated that PAR-6 may
mediate signals from Rac1/Cdc42 to aPKC by interacting with both proteins and
activating aPKC in a GTP-dependent manner
(
Yamanaka et al., 2001). Since
we observed that, as in the case of MDCK, overexpression of a PAR-6 mutant
lacking aPKC-binding domain also showed effects similar to those induced by
aPKCKn during wound healing of MTD1A cells (data not shown)
(
Yamanaka et al., 2001), aPKC
is thought to function as a component of the aPKC-PAR complex in promoting
belt-like AJ formation. Here, we also found that an aPKC-binding scaffold
protein with three PDZ domains, PAR-3, is substantially but not completely
recruited into the premature spot-like AJ complex induced by aPKCλkn,
suggesting that it can translocate to the structure without aPKC activity.
These results indicate that PAR-3 works as a scaffold protein to recruit aPKC
and PAR-6, which can act together to stabilize the complex at the junctional
area. In fact, although indirectly, we observed that PAR-3 is recruited faster
than aPKC into cell-cell contact regions during the normal wound healing
process (
Fig. 1). This is
consistent with the observation that in the C. elegans one-cell
embryo, PAR-3 is transiently present at the cell periphery even in the absence
of either PKC-3 or PAR-6, although both PKC-3 and PAR-6 absolutely require
PAR-3 (
Hung and Kemphues,
1999;
Tabuse et al.,
1998;
Watts et al.,
1996). Considering the fact that the cytoplasmic region of JAM
binds to the first PDZ domain of PAR-3, JAM, which is recruited into the
junctional region as early as ZO-1, may be a membrane target of PAR-3
(
Ebnet et al., 2001;
Itoh et al., 2001,). Then,
aPKC may target PAR-3 anchoring to JAM. We previously suggested the
possibility that aPKC translocates to the junctional region as a ternary
complex with PAR-3 and PAR-6 in MDCKII cells after a calcium switch, since a
considerable amount of the ternary complex was detected even in depolarized
MDCK II cells cultured in low calcium medium
(
Yamanaka et al., 2001). We do
not know the precise reason for the apparent discrepancy between the present
results and the previous data. However, it is possible that the asynchronous
and rapid polarization of MDCK II cells after a calcium switch made it
difficult to detect subtle differences between the recruitment of aPKC and
that of PAR-3. It may also reflect differences between the depolarization
states of cells induced by calcium depletion or mechanical wounding.

In summary, the present work reveals that aPKC is required at the step
where the immature cell-cell junctional complex differentiates into
epithelia-specific asymmetric junctional structures. This finding supports our
hypothesis that the aPKC-PAR system plays an indispensable role in the
establishment of cell polarity by primarily regulating the formation of an
asymmetric submembranous structure to which it anchors. Interestingly, recent
progress in the genetic and molecular analysis of Drosophila or
C. elegans embryos also revealed that epithelial junctions are
assembled in a two-step process, that is, the formation of spot-like AJs along
the lateral membrane rather randomly and their accumulation into the apical
tip of the membrane and development into belt-like AJs
(
Michaux et al., 2001;
Tepass et al., 2001). Many
mutants of polarity proteins including BAZOOKA, a Drosophila
homologue of PAR-3, have been reported to block the transition between these
two steps, indicating that this transition, which is indispensable for
asymmetric membrane domain differentiation is the critical step for epithelial
cell polarization. Many polarity proteins are expected to interact in a
complex fashion during this step. There is no doubt that the identification of
the molecular target of aPKC is one of the important steps to resolve the
molecular basis for this epithelial cell polarization process.

Acknowledgements

We thank S. Tsukita and Y. Takai for providing us with the MTD1- A cell
line and anti-nectin antibody, respectively. This work was supported by grants
from the Japan Society for the Promotion of Science (to S.O.) and the Ministry
of Education, Culture, Sports, Science and Ttechnology of Japan (to S.O. and
A.S.).

Marian Blanca Ramírez from the CSIC in Spain has been studying the effects of LRRK2, a protein associated with Parkinson’s disease, on cell motility. A Travelling Fellowship from Journal of Cell Science allowed her to spend time in Prof Maddy Parson’s lab at King’s College London, learning new cell migration assays and analysing fibroblasts cultured from individuals with Parkinson’s. Read more on her story here.

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